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Abstract

We report a mechanistic analysis of photodamage in coherent anti-Stokes Raman scattering (CARS) microscopy. Photodamage to the myelin sheath in spinal tissues is induced by using the point scan mode and is featured by myelin splitting and shockwaves with broadband emission. Our measurement of photodamage rate versus the excitation power reveals that both linear and nonlinear mechanisms are involved. Moreover, we show that vibrational absorption induced by coherent Raman processes significantly contributes to the nonlinear damage at high peak powers. For CARS imaging of cultured cells, the photodamage is characterized by plasma membrane blebbing and is dominated by a second order mechanism. Our study suggests that for dense samples such as the myelin sheath, CARS imaging induced photodamage can be minimized by using laser beams with relatively long near IR wavelengths and a repetition rate of a few MHz. For less dense samples such as cultured cells, laser pulses of higher repetition rates are preferred.

CARS images of single myelin sheath before and after photodamage induced by point scanning. The x-y imaging and point scanning were carried out at the same condition. (a-b) results with high peak power of the pump (308 W) and Stokes (149 W) beams. f = 0.65 MHz. (e) Intensity trace of point scanning at the position indicated by the red star in (a). (c-d) results with low peak power of the pump (38 W) and Stokes (19 W) beams. f = 15.6 MHz. (f) Intensity trace of point scanning at the position indicated by the red cross in (c). For all images, bar = 5 μm.

Dependence of photodamage rate on the total excitation peak power on the sample. f = 3.9 MHz. The power ratio of pump to Stokes lasers was kept constant at 3.2. The peak power of the pump and Stokes beams were adjusted from 74 W to 277 W and from 23 W to 86 W, respectively.

(a) Dependence of photodamage rate on the pump to Stokes power ratio under the condition of constant CARS signal. f = 3.9 MHz. (b) Dependence of photodamage rate on the repetition rate under the condition of constant CARS signal. The power ratio of pump to Stokes was fixed at 2. f was varied from 0.65 to 39 MHz. To maintain the same CARS signal at different repetition rates, the pump peak power was changed from 286 W to 52 W and the Stokes peak power from 143 W to 26 W accordingly.

Photodamage in CARS imaging of live KB cells. (a) CARS image of a KB cell under normal conditions. (b) CARS image of the same KB cell after 3min of scanning with the pump peak power 373W and Stokes peak power 101W. Bar = 10 μm. (c) Dependence of photodamage rate on the total excitation peak power. f = 7.8 MHz. The power ratio of pump to Stokes lasers was kept at 3.8. The peak power of the pump and Stokes beams were adjusted from 138.4 W to 373 W and from 36 W to 101 W, respectively.